Cystinosis is a metabolic hereditary disease characterized by intracellular accumulation of cystine. Affected individuals typically present with proximal tubulopathy (Fanconi syndrome) before one year of age and without specific treatment progress to end-stage renal failure by the end of the first decade. Cystine accumulation eventually leads to multi-organ dysfunction. The drug cysteamine reduces the intracellular concentration of cystine. However, the need for regularly spaced doses and a number of undesirable side effects render its administration difficult. Moreover, cysteamine does not prevent the proximal renal tubulopathy or the end- stage renal failure. The long-term objective of this project is to develop a kidney-specific gene therapy strategy: retrograde renal vein injection of self-complementary adeno-associated virus (scAAV) expressing a functional CTNS to treat and prevent the proximal tubulopathy and progression of renal defects in cystinosis. As pre-clinical studies, we will use the Ctns-/- murine model for cystinosis. These animals accumulate cystine and cystine crystals in all organs tested and develop kidney injuries similar to those observed in affected humans. Few studies have been performed on AAV for gene delivery in the kidney and most of them used AAV serotype 2 to transduce renal cells in vivo and in vitro. However, the prevalence of neutralizing antibodies in the human population for AAV2 is very high and would probably impact its efficiency for gene delivery. Therefore, in Specific aim 1, we propose to optimize kidney-targeted gene delivery via renal vein injection by testing several AAV serotypes that have the potential of transducing renal cells and a low prevalence of neutralizing antibodies in human. The optimal dose will be also determined. scAAV expressing the green fluorescent protein (GFP) and luciferase reporter genes will be used in this aim and expression will be visualized and quantified using confocal microscopy, flow cytometry and quantitative PCR for GFP, and IVIS imaging system for luciferase. In Specific aim 2, we propose to test this approach based on renal vein injection of scAAV-CTNS as a minimally invasive procedure for treating the renal dysfunction in cystinosis. Renal function will be measured by blood and urine analyses and renal structure by histology. Cystine content and CTNS expression will be measured in the kidney at different time points during a one-year period. The immune response and safety of this directed gene therapy will also be tested. This work represents the first gene therapy treatment strategies for cystinosis and builds the foundations for a future clinical trial. It also represents a proof of concept for a kidney-specific therapy for other hereditary nephropathies. PUBLIC HEALTH RELEVANCE: Cystinosis is an hereditary disease of children characterized by the accumulation of cystine in all the cells of the body leading to cell death and tissue damage including end stage renal failure. The long-term objective of this project is to develop a kidney-specific gene therapy to introduce a functional CTNS gene using a viral vector injected via the renal vein of the patients. As pre-clinical studies, we will use the mouse model for cystinosis that accumulates cystine in all the tissues and develops similar defects including kidney injuries to those of the human children.